The body's pathogen detection system is critical to host defense. Toll-like receptors (TLRs) bind pathogen- associated molecules and activate a network of transcription factors (TFs) that regulate host defense genes. The TFs IRF3, IRF5 and IRF7 are central to TLR signaling in response to viruses and intracellular pathogens. Despite common DNA binding specificities and activation patterns, IRF3, IRF5 and IRF7 (hereafter IRF3/5/7) perform distinct yet overlapping roles in host defense. Much is known regarding differences in the signaling events upstream of IRF3/5/7 activation; however, little is known regarding how IRF3/5/7 discriminate their individual target genes to elicit different biological outcomes and tailor the immune response to pathogens. This proposal integrates multiple genome-scale techniques to examine the mechanisms of specificity and the differential role of IRF3/5/7 in the TLR signaling pathways central to host defense. Genome-wide binding of IRF3/5/7 will be mapped in human macrophages in response to multiple TLR signals activating (dsRNA mimic for TLR3; lipopolysaccharide(LPS) for TLR4; and ssRNA mimic for TLR7). This will provide the first direct comparison of IRF3/5/7 binding in response to TLR signaling. To relate TF binding with functional changes, chromatin accessibility and gene expression will be monitored by DNase I hypersensitivity analysis (DNase- seq) and RNA-seq, respectively. To address the role of DNA binding in IRF3/5/7-specific function, protein- binding microarrays (PBMs) will be used to characterize the DNA binding of IRF3/5/7 dimers. PBM binding data will be integrated with the other genomic datasets to construct a genome-wide model of IRF3/5/7 function. Hypotheses will be tested using cell-based reporter assays (Aim 1). To examine the role of NF-?B - a known cofactor of IRF3/5/7 - we examine IRF binding, chromatin accessibility and gene expression in NF-?B knockout macrophages, and use PBMs to examine DNA binding specificity of IRF-NF-?B complexes. We hypothesize that differential interactions between the IRFs and NF-?B will contribute to their individual biological roles (Aim 2). To examine the role o macrophage master regulators IRF8 and PU.1, we will characterize the genomic binding of IRF8 and PU.1, and integrate this data with maps of IRF binding, chromatin accessibility and gene expression in IRF8 knockout macrophages. To address the role of protein interactions, PBMs will be used to characterize the DNA binding of IRF3/5/7 with the IRF8:PU.1 complex. We hypothesize that cooperative interactions between IRF3/5/7 and IRF8:PU.1 at specific regulatory elements function to tailor IRF-specific functions in a monocyte/macrophage-specific manner. These studies are expected to provide critical insights into the mechanisms that govern IRF3/5/7-specific functions. Our goal is to develop a comprehensive model of the IRF regulatory network that can be used in different cellular contexts, and which may provide insight into autoimmune diseases, such as systemic lupus erythematosus (SLE), which are associated with upregulation of IRF5/7.